Chemotaxis is critical for many developmental and homeostatic processes including neural development, immune response and angiogenesis. Studies in Dictyostelium have improved our understanding of the signaling network that governs chemotaxis. In this system and in mammalian neutrophils, it is clear that phosphoinositide signaling mediates directional responses to chemical cues, but many specific aspects of this network remain unknown. To further analyze this system, I propose to (1) Determine whether inverted localization of phosphoinositide signaling is sufficient to reverse directional migration, and (2) Screen for and characterize temperature-sensitive mutants affecting chemotaxis. In Aim 1, chimeric constructs will be created to target PIPS production to ectopic sites and the localization and activity of these heterologous proteins will be analyzed in cells deficient for PI3K and PTEN. In Aim 2, an alkylating agent will be used to mutagenize Dictyostelium cells and mutants will be screened for defects during chemotaxis. Complementation of these mutant phenotypes using available cDNA and genomic libraries will allow the molecular identification of the responsible gene, and using molecular and biochemical assays, their involvement in directional migration will be analyzed. These experiments are aimed at further characterizing phosphoinositide signaling, as well as identifying new regulatory signals, during chemotaxis. [unreadable] [unreadable] [unreadable]